WestminsterResearchhttp://www.westminster.ac.uk/westminsterresearch

Laccase-assisted approach to graft multifunctional materials of

interest: keratin-EC based novel composites and their

characterisation

Iqbal H.M.N, Kyazze Godfrey, Tron Thierry and Keshavarz T.

This is the peer reviewed version of the following article: Iqbal H.M.N, Kyazze Godfrey,

Tron Thierry and Keshavarz T. (2015) Laccase-assisted approach to graft multifunctional

materials of interest: keratin-EC based novel composites and their characterisation

Macromolecular Materials and Engineering 300 (7) 712-720 1439-2054 , which has been

published in final form at https://dx.doi.org/10.1002/mame.201500003. This article may

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DOI: 10.1002/marc.((insert number)) ((or ppap., mabi., macp., mame., mren., mats.))

Full Paper

Laccase-assisted approach to graft multifunctional materials of interest:

keratin-EC based novel composites and their characterisation

Hafiz M.N. Iqbal1* Godfrey Kyazze1, Thierry Tron2 and Tajalli Keshavarz1*

–––––––––

1 Applied Biotechnology Research Group, Department of Life Sciences, Faculty of Science

and Technology, University of Westminster London, W1W 6UW, United Kingdom

2 Aix Marseille Université, CNRS, Centrale Marseille, iSm2 UMR 7313, 13397, Marseille,

France; *Corresponding author: Tel.: +44 020 79115030; fax: +44 020 79115087; E-mail

addresses: hafiz.iqbal@my.westminster.ac.uk (Hafiz M.N. Iqbal) and

t.keshavarz@westminster.ac.uk (Tajalli Keshavarz)

–––––––––

This study focuses on the evaluation of raw keratin as a potential material to develop

composites with novel characteristics. Herein, we report a mild and eco-friendly fabrication of

in-house extracted feather keratin-based novel enzyme assisted composites consisting of ethyl

cellulose (EC) as a backbone material. A range of composites between keratin and EC using

different keratin: EC ratios were prepared and characterised. Comparing keratin to the

composites, the FT-IR peak at 1,630 cm-1 shifted to a lower wavenumber of 1,610 cm-1 in

keratin-EC which typically indicates the involvement of β-sheet structures of the keratin

during the graft formation process. SEM analysis revealed that the uniform dispersion of the

keratin increases the area of keratin-EC contact which further contributes to the efficient

functionality of the resulting composites. In comparison to the pristine keratin and EC, a clear

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shift in the XRD peaks was also observed at the specific region of 2-Theta values of keratin-

g-EC. The thermo- mechanical properties of the composites reached their highest levels in

comparison to the keratin which was too fragile to be measured for its mechanical properties.

Considerable improvement in the water contact angle and surface tension properties was also

recorded.

FIGURE FOR ToC - ABSTRACT

1. Introduction

Green chemistry needs to overcome many challenges for successful implementation of

innovative technologies to accomplish pollution prevention that reduces and/or eliminates the

consumption and/or generation of harmful waste materials during the entire production

process. In this regard, ever increasing environmental awareness and the demand for

sustainable technology have gained substantial consideration by the academia and industry to

develop an eco-friendly processing designs.[1,2] To address the aforementioned concerns of

global dependence on petroleum-based resources, attention has been directed to the

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engineering of composite materials for targeted applications in different industries.[1-4]

Research on several proteins, including collagen, fibroin, keratin, and others is in progress for

the development of naturally-derived materials. Among the natural materials, keratinous

proteins are interesting candidates to prepare keratin-based composites which have a potential

for utilisation in a variety of bio- and non-bio-sectors. A large variety of fibrous keratins are

available in the form of feather, hair, nail, and horn as bio-waste. These keratin-rich sources

are difficult to degrade as the polypeptide in their structure is tightly packed in α-helix (α-

keratin) or β-sheet (β-keratin) into super coiled chains which are strongly stabilised by several

hydrogen bonds and hydrophobic interactions, in addition to the di-sulphide bonds.[5, 6]

Keratin-based chicken feathers from butchery account for more than 5 million tons per year

worldwide in the form of waste material.[7, 8] Apart from its minor usage in low grade

products such as glue, corrugated paper, cardboard, animal feed and fertilisers etc., the landfill

disposal of poultry feather poses a significant ecological and environmental threat. On the

other hand, from economic and environmental point of view it is also desirable to establish an

effective process for the application of such natural sources.[9] Keratin extracted from feathers

are small proteins, uniform in size, with a molecular weight between 11-65 kDa.[10,11] The

presence of multi-functional groups in keratin, such as di-sulphide, amino, thiol, phenolic and

carboxylic, make it reactive under appropriate reaction conditions. Under reducing

environment, the amino and other groups mentioned above in keratin make its surface

positive, and thus solubilisation takes place.[12] With unique properties of bio-degradability

and non-toxic nature, keratin is among versatile biopolymers that can be modified and

developed into various products of interests. After modification, keratin composites may find

potential applications in bio-medical, pharmaceutical, tissue engineering, and cosmetic

industries. [12]

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In recent years, much effort has been made to fabricate keratin with other suitable materials.

So far most of the work reported deals with blending or grafting of keratin with non-

biodegradable synthetic polymers, such as polyethylene (PE), polypropylene (PP),

polystyrene, and poly (vinyl chloride).[13-19] To overcome the above said problems many

researchers have been proposed to valorise keratinous sources, either through surface grafting

or by blending, to prepare innovative graft composites.[7, 10-12] Grafting is preferred over other

physiochemical techniques in order to obtain the requisite properties which individual

materials fails to demonstrate on their own. Effective modifications should include changes in

chemical group functionality, surface charge, biocompatibility and biodegradability.[20]

Enzymatic grafting is quite a new and interesting technique where the enzyme, as an active

starting material, offers mild and safe reaction conditions to the current practices in the

grafting methods.[2, 20, 21] On the other hand, with respect to health and safety issues linked

with other chemical-based procedures, enzymes offer the potential of eliminating the hazards

associated with reactive reagents. Moreover, there are several benefits for the use of enzymes

in polymeric-based materials synthesis and modifications.[22, 23] Thus, notable amount of

information on the characteristics and hydrolysis of keratin has become available where

recalcitrant keratinous waste are converted into valuable products.[7, 10, 12] The knowledge on

keratin-rich wastes has been robustly increased and their use in cosmetics or in medicines to

enhance drug delivery, and production of biodegradable films, are amongst the outstanding

and emerging biotechnological and biomedical applications. [23, 24]

This study focuses on the evaluation of raw keratin as a potential material to develop

composites with novel characteristics. To the best of our knowledge, in recent literature,

laccase-assisted grafting of keratin material onto EC backbone without addition of any

plasticiser and/or compatibiliser is not reported. In this work we report that newly grafted

composites of keratin and EC with their unique structures under enzymatic environment,

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exhibit novel characteristics such as good thermal stability, tensile strength, and

hydrophobic/hydrophilic balance.

2. Experimental Section

2.1. Chemicals/reagents

In the present study, a fungal laccase from Trametes versicolor with a unit activity of ≥ 10

U/mg was purchased from Sigma-Aldrich Company Ltd., UK and used as received under

standard conditions for grafting purposes. All other chemicals and/or reagents used in the

current work were of analytical grade and purchased from DIFCO (BD UK Ltd., Oxford, UK),

Sigma-Aldrich Company Ltd., UK, and VWR Chemicals (Leicestershire, UK). EC was

obtained from Sigma-Aldrich Company Ltd., UK and used as received according to the

material safety data sheet provided by the company. Chicken feathers were kindly provided

by the Department of Bioengineering, Faculty of Engineering, Ege University, Izmir, Turkey.

All of the chemicals and/or reagents were used as received without further purification unless

otherwise stated.

2.2. Preliminary processing of the chicken feathers

The chicken feathers were washed three times with hot water and ethylene alcohol to avoid

any microbial/dust contamination. Then, they were dried at 60 oC and cut into small

fragments with lengths of 1–2 mm. Clean feathers (10 g) were dissolved in an aqueous

solution of 3% wt. NaOH at 50 oC for 24 h. After the stipulated time the keratin hydrolysate

was filtered in order to separate the insoluble parts of feathers, and then was dialysed. After

48 h of dialysis, 2N HCl was added to the keratin hydrolysate in order to extract the keratin in

the form of precipitate. The keratin solution was centrifuged at 4000 × g for 10 min, and

keratin was washed with distilled water and dried by lyophilisation at -47 °C and at a pressure

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of 133 mbar until fully dried. The dried keratin powder was then stored in a desiccated jar to

avoid free moisture until used in the subsequent graft synthesis experiments.

2.3. “One-pot” synthesis of composites

Previously extracted keratin from chicken feathers and EC were used to prepare composites

with different keratin to EC ratios i.e., keratin: EC; 0:100, 25:75, 50:50, 75:25 and 100:0,

respectively. Briefly, the reaction mixture comprised of keratin and EC was prepared using

laccase and sodium malonate buffer of pH 4.0 followed by incubation at 120 rpm for 30 min

at 25 °C. The above laccase treated mixture was then poured into a sterile labelled petri plate

followed by incubation at 50 oC for 24 h. The newly developed composites were then

removed from their respective casting surfaces (petri plate) and designated as keratin-g-ECA

(prepared using laccase with keratin: EC, 100:0), keratin-g-ECB (prepared using laccase with

keratin: EC, 75:25), keratin-g-ECC (prepared using laccase with keratin: EC, 50:50), keratin-

g-ECD (prepared using laccase with keratin: EC, 25:75), and keratin-g-ECE (prepared using

laccase with keratin: EC, 0:100). All of the prepare composites were then characterised using

a variety of analytical and imaging techniques as described in the section 2.4.

2.4. Characterisation of composites

2.4.1. Fourier transform infrared spectroscopy (FT-IR)

A Perkin-Elmer System 2000 FT-IR spectrophotometer was used to record the infrared

absorption spectra of the grafted composites and their individual counterparts from the

wavelength region of 4000-400 cm-1. All spectra were collected with 64 scans and 2 cm-1

resolution and assigned peak numbers.

2.4.2. Scanning electron microscopy (SEM)

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Scanning electron microscope (Philips, XL-30, FEG SEM; EFI, Netherlands) was used to

analyse the surface morphologies of the grafted composites and their individual counterparts

in ultra-high vacuum mode at an accelerating voltage of 5 kV. The test composites were

prepared using 8 mm diameter aluminium stubs, gold coated for 2 min using the gold

spluttering device and then high definition images were recorded.

2.4.3. X-ray diffraction (XRD)

The test composites and their individual counterparts were scanned at 2-Theta values ranging

from 10° to 100° with a scan speed of 5 °C/min, using thin film attachment, on a Brüker D-8

Advance X-ray diffractometer equipped with Ni filtered Cu K radiation. An updated

MDI/JADE6 software package attached to the Brüker D-8 Advance XRD instrument was

used to record the typical XRD diffractograms.

2.4.4. Differential scanning calorimetry (DSC)

To measure the thermal characteristics of the grafted composites and their individual

counterparts a Pyris Diamond Differential scanning calorimeter (Perkin-Elmer Instruments,

USA) was used. Prior to analyses, test samples were encapsulated in standard aluminium

pans to avoid contamination of the ampoules and the thermal profiles were determined using

DSC~3-7 mg of sample weight. The temperature range was from -50 oC to 250 oC with the

scanning rate of 20 oC/min under nitrogenous atmosphere. DSC analyses were performed in

triplicate and results are presented as mean ± S.E. (standard error) of means (n = 3).

2.4.5. Dynamic Mechanical Analyser (DMA)

The mechanical features in terms of Young’s modulus, tensile strength and elongation at

break point of the composites and individual counterparts were evaluated using a Perkin–

Elmer Dynamic Mechanical Analyser. The test composites were cut into a rectangular shape

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with 8 mm × 4 mm × 0.25 mm dimensions. The crosshead speed was set at a constant tensile

rate of 200 mN min-1 with a total range of 1–6000 mN. DMA analyses were performed in

triplicate and results are presented as mean ± S.E. (standard error) of means (n = 3).

2.4.6. Water contact angle (WCA)

The hydrophobic/hydrophilic characteristics of the composites and individual counterparts

were measured using pendant drop method on a KSV Cam 200 optical contact angle analyser

(KSV instruments Ltd., Finland). A Windows-based KSV-Cam software was used to capture

the images. Ten independent determinations at different sites of the samples were averaged.

3. Results and Discussion

3.1. Fourier transform infrared spectroscopy (FT-IR)

Figure 1 shows a typical FT-IR spectra of the pure keratin and keratin-EC based composites.

The FT-IR spectra shows a peak at 1,630 cm-1 and 1,610 cm-1 for amide I, 1,530 cm-1 with a

shoulder at about 1,520 cm-1 for amide II, and 1,235 cm-1 for amide III, respectively. In

particular, the FT-IR spectra (Fig. 1) shows a predominant absorption band at 1,630 cm-1 and

1,610 cm-1 in case of pure keratin and keratin-EC based composites which typically indicates

the involvement of β-sheet structures from the keratin. Based on the literature data, the peak

of 1,650 cm-1 indicated α-helix structure and the range of 1,631-1,515 cm-1 were related to β-

sheet structure.[10, 25] Figure 1 illustrates an appearance of new peak at 1,717 cm-1 in the

grafted composites. Interestingly, relative to the keratin contents in the graft composites a

decrease in the intensities of the absorption bands at 1,630 cm-1, 1,610 cm-1, 1,530 cm-1, and

1,235 cm-1 was recorded as expected. Evidently, a typical peak at 1,050-1,100 cm-1 region is

due to the C–O–C stretching which designated to the EC molecules.[26] Furthermore, a high

intensity peak at 3,358 cm-1 in the graft composite was observed which is linked to the

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hydrogen-bonded groups at that distinct band region.[10] The peaks at 2,930 and 2,850 cm-1

are characteristic IR bands of aliphatic hydrocarbons of methylene asymmetric C–H

stretching and symmetric C–H stretching, respectively. In addition, the positions of these

bands indicate the conformations of the protein materials: 1,650 cm-1 (α-helix) 1,630 cm-1 (β-

sheet) for amide I, 1,544 cm-1 (α-helix), 1,530 cm-1 (random coil) and 1,520 cm-1 (β-sheet) for

amide II, and 1,230 cm-1 (random coil) for amide III).[27] The amino acids chains of keratin

present hanging groups such as: amino, hydroxyl, thiol and carboxyl that under a redox

system can generate free radicals and thus they can easily react with functional groups of the

cellulose.[28] Based on the availability of the functional groups there are different possibilities

to propose a mechanism of graft formation between keratin and EC. However, figure 2 is only

depicting one potential possibility as a tentative schematic mechanism of graft formation

between keratin and EC under laccase-assisted process.

3.2. Scanning electron microscopy (SEM)

The surface morphologies were investigated by SEM, and the respective micrographs are

shown in Figure 3 (A-F). It can be observed that in case of the grafted composites the keratin

appear homogeneously distributed within the EC backbone. Nevertheless, in the case of the

pure keratin film the adhesion seems quite poor in comparison to the keratin film prepared in

the presence of the laccase (Fig. 3) and (Fig. 3B) respectively, where large number of pores

were observed in the pristine keratin film. Similar results with porous appearance were

observed by Selmin et al. [29] during the membrane formation between regenerated keratin

and ceramides (CERs). The presence of these pores suggests poor adhesion between CERs

and keratin. [29] It has also been reported in literature that the graft copolymerisation modified

the surface morphology; physical, chemical, and microstructural characteristics of grafted

materials.[2, 30] Better results were obtained in the grafted composites by increasing EC ratios

as testified by micrographs (Fig. 3D), and (Fig. 3E) where the keratin material seems more

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adherent to the EC backbone with keratin to EC ratios, 50:50 and 25:75 wt. %. With the

addition of EC, the distribution of pores was reduced on the surfaces of the grafted

composites and finally disappeared as compared to the pristine keratin. On the other hand, the

presence of aggregates provides an evidence of the poor dispersion of the material of interests

within the polymeric matrix and this behaviour is a consequence of the surface chemical

modification that confers a non-polar character to the cellulose surface. [31]

3.3. X-ray diffraction (XRD)

Figure 4 illustrate XRD patterns obtained for pure keratin and keratin-g-EC composites (with

keratin: EC, 100:0; 75:25; 50:50; 25:75 and 0:100). The pure keratin showed a very broad

peak at 2-Theta value of 20o, which specifically corresponds to the �-sheet structure.[32] XRD

pattern showed crystallinity in the pure keratin from the substantial 2-Theta peak values with

sharp appearance at about 26o, 31o, 45o and 56 o. In fact, the wider peaks appeared in the

keratin film prepared using laccase in comparison to the peaks at 26o, 31 o 45 o and 56o

obtained for pure keratin which is possibly due to the laccase action. This significant shift in

peaks clearly indicates the regeneration and breakdown of amorphous and some crystalline

domains at that specific region of 2-Theta values, respectively. However, this phenomenon is

more dominant in the case of the graft with keratin: EC ratio 50:50 as compare to the other

grafts. The keratin-g-EC composites with keratin: EC ratios, 75:25, 50:50 and 25:75 showed a

significant peak shift between 20o to 25o (2-Theta). This is due to the occurrence of strong

intermolecular and intramolecular bonding interactions which are typically indicate the

involvement of β-sheet structures from the keratin as evident by the FT-IR spectra. If there

was no or weak interaction between keratin and EC molecules in the graft, each component

would have its own individual region and the XRD patterns would be expressed as the simple

mixture of keratin and EC. On the other hand, EC had broader diffraction with a broad peak at

20o in the XRD-patterns that represents the highly amorphous nature of EC.[4, 33]

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3.4. Differential Scanning Calorimetry (DSC)

Thermal properties such as glass transition temperature (Tg), melting temperature (Tm),

crystallisation temperature (Tc) and melting enthalpy (∆Hm J/g) obtained from the DSC

studies are summarised in Table 1. For each specimen an endo-thermal peak was observed

during the temperature scan. The first endothermic peak for pure keratin and keratin only

film was visible at low temperature about 42 °C and 34 °C, respectively, which is in turn

associated with the amount of hydrogen-bound molecules (sometimes referred to as the

denaturation peak or glass transition temperature). The results showed a slight change in the

glass transition temperature (Tg) from 42 oC to 34 oC and melting temperature (Tm) from 232

oC to 210 oC of the keratin film prepared using laccase as compared to the pristine keratin

film. Based on the data reported in the literature Tm of the polymers is linked to the size of

the polymers and/or dependent on the crystalline domains of the polymers.[34] This initial

reduction in the Tm of the keratin film prepared using laccase is an indication of the clear

change or disruption in the size of the crystalline domains during the reaction phase. The

DSC results are well in support with the observations recorded through XRD studies where

the wider peaks appeared in the graft composites as compare to the sharp peaks of the pure

keratin at 26o, 31o, 45o and 56o. However, the introduction of the keratin into the EC

determines a shift of the melting temperatures to higher values, indicating an increase of the

thermal stability. On high temperature, keratin can contribute to the formation of a protective

barrier, on the material surface. This layer delays the heat transfer to the system and

diminishes the release of the gaseous products from the system.[35]

3.5. Dynamic Mechanical Analyser (DMA)

Table 2 provides the results of mechanical characteristics of the composites and individual

counterparts. The keratin film prepared using laccase was too fragile to permit measurement

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of its mechanical characteristics compared to the pure untreated keratin film. However, the

composite film prepared using keratin and EC with keratin: EC ratio 75:25 was fairly flexible

and strong judged by the tensile strength (22 MPa), elongation at break (37%) and Young’s

modulus (1.1 GPa). The further increase of EC content ratio resulted in little additional

change in the ultimate strength and elongation. However, an increased Young’s modulus,

suggests that further increase of EC content up to 75% make the film stiffer. As it can be seen

in Table 2, keratin film incorporated with 50% wt. EC showed a considerable improvements

in the tensile strength (55 MPa), elongation (18%) and Young’s modulus (2.4 GPa) in

comparison to the pristine keratin. From these results, laccase-assisted grafting of keratin and

EC was found to increase mainly the strength and flexibility of the keratin. Based on the

literature, appropriate addition of polymers like cellulose, chitosan and glycerol, could control

the mechanical properties of keratin films and keratin containing composites.[36] As shown in

Table 2, the grafted composites with the “higher” keratin (obtained from chicken feathers)

have lower tensile strength. This has also been reported by other researchers,[34] and can be

ascribed to the fact that the strength of hydrolysed chicken feathers is insufficient to test for

mechanical characteristics. Our tests showed that laccase-assisted grafting brings a slight

increase in the tensile strength in the resulting composites relative to the pristine keratin.

Recently, it has been reported that laccase treatments can significantly improve the existing

and/or impart new physicochemical characteristics including dry and wet tensile strength

through various cross-linking reactions.[2, 10, 37]

3.6. Water contact angle (WCA)

The hydrophobic/hydrophilic characteristics in terms of water contact angle and the surface

tension properties of the newly synthesised graft composites and their individual counterparts

are presented in Figure 5. The water contact angle and surface tension values for the pure

keratin film prepared in the absence of laccase were 61o and 42 mN/m, respectively, while in

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comparison to this an increase in the water contact angle from 61o to 70o and surface tension

from 42 mN/m to 48 mN/m was observed for the keratin film prepared in the presence of

laccase (Fig. 5). This initial reduction in the hydrophilicity of the pure keratin film is probably

because of the laccase action. The water contact angle values for the graft composites prepared

with different keratin to EC ratios i.e., keratin: EC; 100:0, 75:25, 50:50, 25:75 and 0:100 were

25o, 70o, 48o, 52o, 49o and 47o respectively. Hence, with the increase of EC content ratio

indicates that the hydrophilic property of keratin-g-EC composites is much better than that of

the pure keratin. It can be expected that the keratin-g-EC composite with high hydrophilicity is

more suitable for cell adhesion and proliferation than pure keratin film prepared directly from

the keratin hydrolysate. It has also been reported in literature that the hydrophilic/hydrophobic

balances are among critical factors which affects the platelet adhesion potential and the

cytocompatibility features of the materials, and these properties would endow the polymeric

materials as a potential candidate for various biomedical type applications.[2, 10]

4. Conclusions

In this study, in-house extracted keratin from waste chicken feathers in combination with EC

was used to develop novel composites using different keratin to EC ratios. According to the

results obtained and discussed above, the following conclusions can be drawn:

1) A novel laccase-assisted method was developed to manufacture keratin-EC based novel

composites.

2) The technology developed in the present investigation enables the re-use of the chicken

feather, a very troublesome waste of the poultry industry, which contributes a major

role in the current ecological and/or environmental pollution problems.

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3) As evidenced by characterisation analyses, considerable improvement in the

morphology, thermo-mechanical and wettability features was recorded in the newly

synthesised composites that individual material (keratin) fail to demonstrate on its own.

4) The main benefits of laccase-assisted grafting processes are the facile synthesis in the

absence of harmful solvents and chemicals, the mild eco-friendly and energy saving

reaction conditions.

Acknowledgements: The financial support provided by the University of Westminster

London UK under the Cavendish Research Scholarship program is thankfully acknowledged.

The authors are thankful to the Drs. Jonathan C. Knowles, George Georgiou and Nicola

Mordan (Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute

(EDI), London, UK) on providing laboratory facilities for characterisation analyses. The

valuable help and support provided by Mr. Farzad Fountain (Division of Biomaterials and

Tissue Engineering, UCL EDI, London, UK) for X-ray diffraction analyses is thankfully

acknowledged.

Received: Month XX, XXXX; Revised: Month XX, XXXX; Published online:

Keywords: Biopolymers, Laccase, Composites, Characterisation

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Figure 1. Typical FT-IR spectra of the pure keratin and keratin-EC based graft composites i.e., keratin-g-ECA, keratin-g-ECB, keratin-g-ECC, keratin-g-ECD, and keratin-g-ECE prepared using laccase as a model catalyst.Where, *: Pure keratin film prepared without laccase treatment

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Figure 2. A tentative schematic representation of proposed mechanism of graft formation between keratin and EC under laccase-assisted environment.

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Figure 3. SEM micrograph of the pure keratin (A) and keratin-EC based graft composites i.e., keratin-g-ECA (B), keratin-g-ECB (C), keratin-g-ECC (D), keratin-g-ECD (E), and keratin-g-ECE (F) prepared using laccase with different keratin to EC ratios.

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Figure 4. Typical X-ray diffracogram of the pure keratin (A) and keratin-EC based graft composites i.e., keratin-g-ECA (B), keratin-g-ECB (C), keratin-g-ECC (D), keratin-g-ECD (E), and keratin-g-ECE (F) prepared using laccase with different keratin to EC ratios.

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Figure 5. Water contact angle (bars in colour) and surface tension (red line) measurements of the pure keratin and keratin-EC based graft composites i.e., keratin-g-ECA, keratin-g-ECB, keratin-g-ECC, keratin-g-ECD, and keratin-g-ECE prepared using laccase with different keratin to EC ratios.Where, *: Pure keratin film prepared without laccase treatment

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Table 1. DSC-thermal characteristics of the individual polymers i.e., keratin and EC and their grafted composites i.e., keratin-g-ECA, keratin-g-ECB, keratin-g-ECC, keratin-g-ECD, and keratin-g-ECE prepared using laccase with different keratin to EC ratios.

Sample ID Tg (°C) Tm (°C) Tc (°C) ∆Hm (J/g)

Keratina 42±0.35 232±2.22 112±1.50 31±1.25

Keratin-g-ECA 34±0.31 210±1.22 177±1.35 24±0.35

Keratin-g-ECB 74±0.20 228±2.65 165±2.10 16±0.08

Keratin-g-ECC 105±0.95 223±1.10 169±1.85 9.5±0.45

Keratin-g-ECD 88±1.10 212±2.20 155±2.21 12±0.12

Keratin-g-ECE b 199±1.65 120±1.32 92±1.11

a) Pure keratin film prepared without laccase treatment; b) not detected.

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Table 2. DMA-mechanical characteristics of the individual polymers i.e., keratin and EC and their grafted composites i.e., keratin-g-ECA, keratin-g-ECB, keratin-g-ECC, keratin-g-ECD, and keratin-g-ECE prepared using laccase with different keratin to EC ratios.

Sample IDTensile strength

(MPa)

Young’s modulus

(GPa)

Elongation at break

(%)

Keratina b b b

Keratin-g-ECA b b b

Keratin-g-ECB 22±0.09 1.1±0.31 37±0.33

Keratin-g-ECC 55±0.15 2.4±0.42 18±0.45

Keratin-g-ECD 91±0.21 3.1±0.25 8±0.28

Keratin-g-ECE 122±0.42 3.5±0.65 9±0.15

a) Pure keratin film prepared without laccase treatment; b) not detected.

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Table of Contents

This study focuses on the evaluation of raw keratin as a potential material to develop

composites with novel characteristics. Herein, we report a mild and eco-friendly fabrication of

in-house extracted feather keratin-based novel composites consisting of ethyl cellulose as a

backbone material.